Histidylated poly(lysine) highly branched polymers for siRNA delivery

There has been a great interest in the research into the therapeutic applications of gene silencing in humans. This stems from the ability of small interfering RNA (siRNA, RNA fragments of 21-23 nucleotides) to induce RNA interference (RNAi), a pathway in eukaryotic cells that leads to destruction of complementary mRNA. The RNAi pathway can be used to silence endogenous genes that can cause diseases such as cancer. Silencing or knocking down of genes is also possible for the genes that are necessary for the proliferation of organisms that cause infectious diseases, and thus this approach of achieving gene silencing can be used for disease prevention or treatment. Gene silencing can be induced in any tissue, in theory, and knocking down genes has been reported to be achieved in diseases such as liver cirrhosis, hepatitis B (HBV), and ovarian and bone cancers. This effect can last from few days in rapidly dividing cells to several weeks in non-dividing cells and thus requires repeated administration. To date, developing safe and effective delivery systems for siRNA in vivo that can achieve intracellular bioavailability is the main challenge for achieving a widespread use of RNAi for disease prevention and treatment. The delivery of siRNA can be achieved either locally or systemically. siRNA formulation strategies are often based on particulates associated approaches (e.g. lipid or polymer-based colloids). If delivered systemically, most siRNA formulations tend to accumulate in organs such as liver, lungs, or kidneys (organs of the reticuloendothelial system). To target other organs either local delivery can be used (such as to the eye) or via the lymphatic system. A key property of siRNA delivery vehicles, in addition to promoting the delivery of siRNA into the cells, is avoiding inducing immune responses. Thus, approaches that result in obtaining vehicles that minimise the activation of the immune system is required.
In this work we propose to develop charge-neutral particles by first examining cationic lipid and polymeric excipients to complex model siRNA. Pre-complexation of siRNA by charge-charge interactions can result in particulates designed to dissociate intracellularly. The complexed siRNA can be further formulated for lymphatic uptake via subcutaneous administration and for local use within the eye (intraocular). Initial efforts will utilise lysine-histidine amino acid-based hyperbranched polymers for siRNA, which would reduce cytotoxicity. We will also utilise state of the art characterisation techniques including dynamic light scattering (DLS), Nanoparticle Tracking Analysis (NTA) and electron microscopy to assess the particle size and size distribution as this is key in particle uptake by the cells, flow cytometry and in vitro cell assays for functionality testing. Characterisation will allow correlations to be determined to optimise formulation strategies by an iterative process. The work will involve the following stages, which align with the CDT core themes of: Advanced Product Design, Pharmaceutical Process Engineering and Complex Product Characterisation:
- Formulation of hyperbranched polymer-siRNA particles: This will require determining the optimum ratio of polymer to siRNA ratio. Isothermal titration calorimetry will be utilised in this stage to study the mechanism of particle assembly, formation, and binding properties.
- Characterisation of particles: This includes particle size analysis (DLS, NTA), surface properties characterisation.
- In-vitro studies: cell culture and transfection study with suitable assays to determine nanoparticle uptake and cell viability.
- In-vivo studies. The project will end with a short in-vivo study, looking to explore the potency of the lead formulation in a suitable mouse model.

Pharmaceutical technologies underpin healthcare product development. Medicinal products are becoming increasingly complex, and while the next generation of research scientists in the life- and pharmaceutical sciences will require high competency in at least one scientific discipline, they will also need to be trained differently than the current generation. Future research leaders need to be equipped with the skills required to lead innovation and change, and to work in, and connect concepts across diverse scientific disciplines and environments. This CDT will train PhD scientists in cross-disciplinary areas central to the pharmaceutical, healthcare and life sciences sectors, whilst generating impactful research in these fields. The CDT outputs will benefit the pharmaceutical and healthcare sectors and will underpin EPSRC call priorities in the development of low molecular weight molecules and biologics into high value products.

Benefits of cohort research training: The CDT's most direct beneficiaries will be the graduates themselves. They will develop cross-disciplinary scientific knowledge and expertise, and receive comprehensive soft skills training. This will render them highly employable in R&D in the pharmaceutical, healthcare and wider life-sciences sectors, as is evidenced by the employment record in R&D intensive jobs of graduates from our predecessor CDTs. Our students will graduate into a supportive network of alumni, academic, and industrial scientists, aiding them to advance their professional careers.

Benefits to industry: The pharmaceutical sector is a key part of the UK economy, and for its future success and international competitiveness a skilled workforce is needed. In particular, it urgently needs scientists trained to develop medicines from emerging classes of advanced active molecules, which have formulation requirements that are very different from current drugs. The CDT will make a considerable impact by delivering a highly educated and skilled cohort of PhD graduates. Our industrial partners include big pharma, SMEs, CROs, CMOs, CMDOs and start-up incubators, ensuring that CDT training is informed by, and our students exposed to research drivers in, a wide cross-section of industry. Research projects in the CDT will be designed through a collaborative industry-academia innovation process, bringing direct benefits to the companies involved, and will help to accelerate adoption of new science and approaches in the medicines development. Benefit to industry will also be though potential generation of IP-protected inventions in e.g. formulation materials and/or excipients with specific functionalities, new classes of drug carriers/formulations or new in vitro disease models. Both universities have proven track records in IP generation and exploitation. Given the value added by the pharma industry to the UK economy ('development and manufacture of pharmaceuticals', contributes £15.7bn in GVA to the UK economy, and supports ~312,000 jobs), the economic impacts of high-level PhD training in this area are manifest.

Benefits to society: The CDT's research into the development of new medical products will, in the longer term, deliver potent new therapies for patients globally. In particular, the ability to translate new active molecules into medicines will realise their potential to transform patient treatments for a wide spectrum of diseases including those that are increasing in prevalence in our ageing population, such as cardiovascular (e.g. hypertension), oncology (e.g. blood cancers), and central nervous system (e.g. Alzheimer's) disorders. These new medicines will also have major economic benefits to the UK. The CDT will furthermore proactively undertake public engagement activities, and will also work with patient groups both to expose the public to our work and to foster excitement in those studying science at school and inspire the next generation of research scientists.